PROJECT SUMMARY Alveolar macrophages (AMs) are lung-resident phagocytes essential to pulmonary host defense. Perturbation of AM biology contributes to a variety of lung diseases (including acute respiratory distress syndrome and ventilator-induced lung injury) and to susceptibility to respiratory infections. Detailing the molecular mechanisms that control AM activation and development will therefore inform new therapeutic strategies to modulate pulmonary inflammation in multiple diseases. During the course of our initial award, we discovered that mice lacking the actin-bundling protein L-plastin (LPL; LPL-/- mice) were profoundly susceptible to pneumococcal lung infection. We discovered that susceptibility to lung infection correlated with a deficit of AMs, and we confirmed an AM-specific requirement for LPL in bacterial clearance using CD11c.Cre-specific deletion of LPL (newly generated CD11.Cre+-LPLfl/fl mice). We then leveraged our expertise and reagents to probe a novel and significant finding that AM development specifically requires LPL (Blood, 2016). This renewal is built upon published and preliminary data generated while investigating the role of LPL in AM development and function. First, we found that LPL is specifically required for the migration of AM precursor cells (monocytes and/or pre-AMs) into the alveolar space, where the essential growth factor GM-CSF is produced. Second, we found that LPL enables the migration of monocytes and macrophages by supporting podosomes. Podosomes are integrin-mediated, F-actin-based organelles that promote macrophage adhesion and migration. Podosomes also mediate mechanotransduction, translating mechanical force exerted upon the cell into intracellular signaling that alters macrophage biology. Third, we found that AMs from LPL-/- mice exhibited defective IL-1? production after NLRP3 inflammasome activation, and that NLRP3 inflammasome activation was mechanosensitive. Finally, we found that airway administration of exogenous GM-CSF to neonatal LPL-/- mice, during the normal temporal window of AM development, rescued AM numbers and protected adult animals from subsequent bacterial infection. From these findings, we formulated our central hypothesis: LPL, via its function in podosomes, mediates mechanotransduction that regulates macrophage pro-inflammatory signaling. We will test this hypothesis by 1) defining, in unprecedented detail, the formation of podosomes in the presence and absence of LPL; 2) determining which macrophage pro- and anti-inflammatory states require LPL and/or are mechanosensitive; and 3) defining mechanisms by which GM-CSF regulates AM mechanosensitivity and IL-1? production in WT and LPL-deficient AMs to show that AM biology can be therapeutically modified. The combination of the PI's experience in cell biology, immunology and infectious disease, the assembled team of collaborators, and the environment at Washington University ensure that this work will be accomplished. Completion of these studies will illuminate a novel pathway by which alteration in tissue stiffness (compliance) during disease states may alter the inflammatory environment through macrophage mechanotransduction.